The GJB2 (Cx26) Gene Variants in Patients with Hearing Impairment in the Baikal Lake Region (Russia)

The GJB2 (Cx26) gene pathogenic variants are associated with autosomal recessive deafness type 1A (DFNB1A, OMIM #220290). Direct sequencing of the GJB2 gene among 165 hearing-impaired individuals living in the Baikal Lake region of Russia identified 14 allelic variants: pathogenic/likely pathogenic—nine variants, benign—three variants, unclassified—one variant, and one novel variant. The contribution of the GJB2 gene variants to the etiology of hearing impairment (HI) in the total sample of patients was 15.8% (26 out of 165) and significantly differed in patients of different ethnicity (5.1% in Buryat patients and 28.9% in Russian patients). In patients with DFNB1A (n = 26), HIs were congenital/early onset (92.3%), symmetric (88.5%), sensorineural (100.0%), and variable in severity (moderate—11.6%, severe—26.9% or profound—61.5%). The reconstruction of the SNP haplotypes with three frequent GJB2 pathogenic variants (c.-23+1G>A, c.35delG or c.235delC), in comparison with previously published data, supports a major role of the founder effect in the expansion of the c.-23+1G>A and c.35delG variants around the world. Comparative analysis of the haplotypes with c.235delC revealed one major haplotype G A C T (97.5%) in Eastern Asians (Chinese, Japanese and Korean patients) and two haplotypes, G A C T (71.4%) and G A C C (28.6%), in Northern Asians (Altaians, Buryats and Mongols). The variable structure of the c.235delC-haplotypes in Northern Asians requires more studies to expand our knowledge about the origin of this pathogenic variant.


Introduction
The GJB2 (Cx26) gene (13q12.11, MIM 121011) pathogenic variants associated with autosomal recessive deafness type 1A (DFNB1A, OMIM #220290) are the main cause of hereditary non-syndromic hearing impairment (HI). The degree of hearing loss ranges from profound congenital deafness to mild, progressive HI manifesting in late childhood, and is highly dependent on certain GJB2 genotypes [1]. At present, more than 560 allelic variants of the GJB2 gene are known (https://www.ncbi.nlm.nih.gov/clinvar/?term=GJB2%5Bgene% 5D, accessed on 20 March 2023). The average contribution of the GJB2 pathogenic variants to the etiology of HI in the world was estimated to be 17.3%. The highest contribution of The DNA samples of 165 patients with HI from 160 unrelated families were collected in 2019. The majority of patients were of Buryat (47.8%; n = 79) and Russian ethnicity (46.1%; n = 76). Patients of other ethnicities accounted for 6.0% (n = 10). The males accounted for 41.2% (n = 68) and females-58.8% (n = 97). The average age was 50.7 ± 15.5 years (Table 1).

Clinical and Audiological Examinations
For each patient, a medical history was collected, including the information on previous illnesses, allergological history, injuries and/or surgeries, the use of ototoxic drugs and the exposure to industrial noise. The hearing thresholds were determined by pure-tone audiometry, using a clinical tonal audiometer "AA222" ("Interacoustics", Middelfart, Denmark), according to the current clinical standards. Air-conduction and bone conduction thresholds were obtained at 0.125, 0.25, 0.5, 1, 2, 4 and 8 kHz. Severity of hearing loss was defined by pure tone average (PTA 0.5,1,2,4kHz ), as mild (25-40 dB), moderate (41-70 dB), severe (71-90 dB) or profound (above 90 dB). The median hearing thresholds in PTA 0.5,1,2,4kHz of GJB2 genotypes were compared using the Mann-Whitney U-test. Differences were considered statistically significant at p < 0.05.

In Silico Pathogenicity Analysis
Analysis of the allelic variants with uncertain clinical significance (VUS) located in noncoding upstream 5 region of the GJB2 gene was performed using the Ensemble centroid instrument generated in the Sfold software 2.2. (https://sfold.wadsworth.org/cgi-bin/ srna.pl, accessed on 15 September 2022).

Brief Information about Studied Region
The Republic of Buryatia includes 21 districts and two cities (Ulan-Ude and Severobaikalsk) (https://egov-buryatia.ru, accessed on 15 September 2022), with an area of 351.3 thousand km 2 . This region of the Russian Federation borders with Mongolia. The population of the Republic of Buryatia is 978,600 people, with an average density of 2.78 people/km 2 . The major ethnic groups are Buryats (30.1%) and Russians (59.4%) (https://burstat.gks.ru/vpn2020, accessed on 25 January 2023). The Buryats are a Mongolicspeaking people and one of the largest indigenous groups in Siberia. Buryats share many customs with other Mongols, including nomadic herding and using portable dwellingsyurts. The majority of the Buryat population lives in the Republic of Buryatia, Irkutsk Oblast' and Zabaykalsky Krai of Russia. Buryats also live in the northeastern part of Mongolia and China (Inner Mongolia).

Ethical Control
All patients gave written informed consent for participation in the study. This study was approved by the local Biomedical Ethics Committee at the Yakut Scientific Center of Complex Medical Problems, Yakutsk, Russia (Yakutsk, protocol No. 50 of 24 December 2019).

Identified Variants in the GJB2 Gene
Large DFNB1 deletions of 309 kb-del(GJB6-D13S1830), 232 kb-del(GJB6-D13S1854) and 101 kb-del(GJB2-d13S175) were not found in our sample. Sequencing of the coding (exon 2) and non-coding (exon 1) with flanking intronic regions of the GJB2 gene in 165 patients with HI from the Republic of Buryatia revealed fourteen allelic variants. In the protein-coding region (exon 2) of the GJB2 gene, eleven variants were identified. In the non-coding regions (intron 1 and 5 UTR region) three variants were identified ( Figure 1). Nine allelic variants are known as pathogenic or likely pathogenic (PLP), three variants are known as benign, one variant c.-254C>T is unclassified (uncertain significance) and one variant c.-49G>A was not described previously ( Figure 1). population of the Republic of Buryatia is 978,600 people, with an average density of 2.78 people/km 2 . The major ethnic groups are Buryats (30.1%) and Russians (59.4%) (https://burstat.gks.ru/vpn2020, accessed on 25 January 2023). The Buryats are a Mongolic-speaking people and one of the largest indigenous groups in Siberia. Buryats share many customs with other Mongols, including nomadic herding and using portable dwellings-yurts. The majority of the Buryat population lives in the Republic of Buryatia, Irkutsk Oblast' and Zabaykalsky Krai of Russia. Buryats also live in the northeastern part of Mongolia and China (Inner Mongolia).

Ethical Control
All patients gave written informed consent for participation in the study. This study was approved by the local Biomedical Ethics Committee at the Yakut Scientific Center of Complex Medical Problems, Yakutsk, Russia (Yakutsk, protocol No. 50 of 24 December 2019).

Identified Variants in the GJB2 Gene
Large DFNB1 deletions of 309 kb-del(GJB6-D13S1830), 232 kb-del(GJB6-D13S1854) and 101 kb-del(GJB2-d13S175) were not found in our sample. Sequencing of the coding (exon 2) and non-coding (exon 1) with flanking intronic regions of the GJB2 gene in 165 patients with HI from the Republic of Buryatia revealed fourteen allelic variants. In the protein-coding region (exon 2) of the GJB2 gene, eleven variants were identified. In the non-coding regions (intron 1 and 5′UTR region) three variants were identified ( Figure 1). Nine allelic variants are known as pathogenic or likely pathogenic (PLP), three variants are known as benign, one variant c.-254C>T is unclassified (uncertain significance) and one variant c.-49G>A was not described previously ( Figure  1).

GJB2 Genotypes in Patients with HI
Eighteen different GJB2 genotypes were identified in patients with HI in the Republic of Buryatia (n = 165). Among them, seven GJB2 genotypes with biallelic pathogenic/likely pathogenic (PLP) variants in compound heterozygous and in homozygous state were found in 26 patients (15.8%) ( Table 2).  GJB2 genotypes with biallelic PLP variants Six different GJB2 genotypes with single recessive pathogenic variant were found in nine (5.5%) patients. Five different GJB2 genotypes with benign and unclassified variants were detected in 32 (19.4%) patients. In the other 98 (59.4%) patients we found no changes in the GJB2 gene sequence ( Table 2). Two heterozygous substitutions c.[-254C>T(;)516G>C] and c.[79G>A(;)341A>G] are presumably in the cis-position, which was previously shown in other studies [31][32][33]40,41]. In one patient with heterozygous c.23C>T p.(Thr8Met) variant, a homozygous c.457G>A p.(Val153Ile) variant was also found ( Table 2). Based on clinical significance of the identified GJB2 variants, the contribution of biallelic PLP variants to the etiology of HI in the total sample of patients in our study was 15.8% (26/165) ( Table 2).

Contribution of the GJB2 Variants to the Etiology of HI in Buryat and Russian Patients
Furthermore, we subdivided our sample of HI patients into two main groups based on their ethnicity ( Figure 2). Contribution of the GJB2 variants to the etiology of HI among Buryat patients was lower-5.1% (4/79), compared to Russian patients-28.9% (22/76).

Contribution of the GJB2 Variants to the Etiology of HI in Buryat and Russian Patients
Furthermore, we subdivided our sample of HI patients into two main groups based on their ethnicity ( Figure 2). Contribution of the GJB2 variants to the etiology of HI among Buryat patients was lower-5.1% (4/79), compared to Russian patients-28.9% (22/76).

Discussion
In the present study, molecular genetic analysis of the non-coding (exon 1) and coding (exon 2) regions of the GJB2 gene was performed on 165 individuals with HI from the Republic of Buryatia (Eastern Siberia, Russia). Among fourteen identified allelic variants, nine variants are known as pathogenic or likely pathogenic, three variants are known as benign, one variant (c.-254C>T) is unclassified and one variant (c.-49G>A) has not been previously described. The total contribution of the GJB2 variants to the etiology of HI in observed patients in the Republic of Buryatia was 15.8% (26 out of 165); however, there were significant differences between two main ethnic groups of patients: 28.9% in Russians and 5.1% in Buryats. The contribution of the GJB2 variants in Russian patients defined in this study (28.9%) corresponds to the data on Russian patients with HI (33.0%) living in neighboring Siberian region (the Sakha Republic) [32]. However, the low contribution of the GJB2 variants to HI in Buryat patients (5.1%) belonging to one of the indigenous Siberian populations, is lower compared to other Siberian ethnic groups (15.1%-in Altaian patients, 22.3%-in Tuvinian patients, and 51.2%-in Yakut patients) [31][32][33]42], while it is similar to the proportion of GJB2-related HI in patients from neighboring Mongolia (4.5-6.9%) [14,43]. It should be noted that the frequency of HI associated GJB2 variants in Mongolia is one of the lowest in the world [1].

Discussion
In the present study, molecular genetic analysis of the non-coding (exon 1) and coding (exon 2) regions of the GJB2 gene was performed on 165 individuals with HI from the Republic of Buryatia (Eastern Siberia, Russia). Among fourteen identified allelic variants, nine variants are known as pathogenic or likely pathogenic, three variants are known as benign, one variant (c.-254C>T) is unclassified and one variant (c.-49G>A) has not been previously described. The total contribution of the GJB2 variants to the etiology of HI in observed patients in the Republic of Buryatia was 15.8% (26 out of 165); however, there were significant differences between two main ethnic groups of patients: 28.9% in Russians and 5.1% in Buryats. The contribution of the GJB2 variants in Russian patients defined in this study (28.9%) corresponds to the data on Russian patients with HI (33.0%) living in neighboring Siberian region (the Sakha Republic) [32]. However, the low contribution of the GJB2 variants to HI in Buryat patients (5.1%) belonging to one of the indigenous Siberian populations, is lower compared to other Siberian ethnic groups (15.1%-in Altaian patients, 22.3%-in Tuvinian patients, and 51.2%-in Yakut patients) [31][32][33]42], while it is similar to the proportion of GJB2-related HI in patients from neighboring Mongolia (4.5-6.9%) [14,43]. It should be noted that the frequency of HI associated GJB2 variants in Mongolia is one of the lowest in the world [1].
[35delG];[35delG] 108 dB in PTA, profound HI) was previously demonstrated by the multicenter genotype-phenotype studies [44,45]. In this regard, the GJB2 genotype c.  (Figure 4). To our knowledge, our earlier study [42] is the only audiological analysis of a large cohort of patients (n = 40) homozygous for the c.-23+1G>A variant. In that study we reported the median PTA 0.5,1.0,2.0,4.0kHz to be 86 dB (severe HI), which is better than that in patients with c.
[35delG];[35delG] genotype (102 dB, profound HI) (Figure 4). Taken together, our findings in patients with combined genotypes c.[-23+1G>A];[-254C>T(;)516G>C] (median 64.3 dB, moderate HI) indicate that these GJB2-variants (c.-23+1G>A and c.516G>C) may be associated with more mild phenotypes than observed with homozygous c.35delG genotypes. It is possible that our sample of patients is biased towards severe and profound HI, and as a result, we did not find more cases with milder phenotypes.
The most interesting results were obtained from the reconstruction of 54 mutant chromosomes with c.235delC using four overlapping SNPs (SNPs 5, 6, 7 and 11) ( Figure 5). Three different c.235delC-haplotypes (G A C T, G A T T and G A C C) were reconstructed. In this regard, we divided the Asian patients with c.235delC in two groups: the Northern Asians (Altaian, Buryat and Mongolian patients) (this study and [7,21]) and the Eastern Asians (Chinese, Korean and Japanese patients) [7] (Figure 6). The G A T T haplotype was previously found in Eastern Asians with low frequency (8.3%, only in one chromosome of Japanese patients) (Table S2, Figure 6). The c.235delC-haplotype G A C T was presented with high frequency in the Eastern Asians (97.5%, CI: 87.1-99.4%) and with lower frequency in the Northern Asians (71.4%, CI: 44.9-88.2%) ( Figure S4). The c.235delC-haplotype G A C C was absent in Eastern Asians (0%, CI: 0-0.8%) and was present only in Northern Asians (28.6%, CI: 11.8-55.1%) (p < 0.05) ( Figure 6, Figure S4). Two main haplotypes, G A C T and G A C C, differ by one distant SNP marker (rs747931,~62.5 kB from c.235delC, underlined).
The diversity of the c.235delC haplotypes in different geographical regions may indicate an independent origin of this GJB2 pathogenic variant in Eastern and Northern Asians. However, all previous research focusing on the analysis of the haplotypes bearing c.235delC suggested the hypothesis on the single origin of c.235delC [7,20,21,23,43,[58][59][60]. Thus, in the study by Yan et al. (2003), seven SNPs flanking c.235delC were analyzed in patients with HI from China, Japan, Korea and Mongolia, and the only one haplotype A G A C (SNP2-V27I-E114G-SNP1) associated with c.235delC was found [7]. In addition, the age of c.235delC (~11,500 years) estimated by one distant SNP marker (SNP6 or rs747931) was calculated [7]. It is worth noting that this particular SNP differentiated the haplotypes found in our study (G A C T and G A C C, underlined) (Table S2, Figure 6). Assuming a probable recombination of this distant SNP (rs747931) and a potential effect of genetic drift, our data on the structure of variable haplotypes in North Asians may be consistent with the hypothesis proposed by Yan et al. (2003) about a single origin of c.235delC in the Baikal Lake region and its spreading to Mongolia, China, Korea and Japan through subsequent migrations [7]. However, in recent studies, a "younger" age of c.235delC was determined:~6500 years and~1125-3150 years in the Japanese and Altaian carriers of c.235delC, respectively [20,21]. These discrepancies can be explained by differences in the methods of the age estimation, the panels of used genetic markers, and the sample sizes in different studies [20,21], although this may be due to different genetic background of populations studied. We have identified the c.235delC haplotype for the first time in the presumed region of c.235delC origin (the Baikal Lake region) and believe that subsequent studies of the structure of haplotypes bearing c.235delC will elucidate the origin of this GJB2 pathogenic variant. The diversity of the c.235delC haplotypes in different geographical regions may indicate an independent origin of this GJB2 pathogenic variant in Eastern and Northern Asians. However, all previous research focusing on the analysis of the haplotypes bearing c.235delC suggested the hypothesis on the single origin of c.235delC [7,20,21,23,43,[58][59][60]. Thus, in the study by Yan et al. (2003), seven SNPs flanking c.235delC were analyzed in patients with HI from China, Japan, Korea and Mongolia, and the only one haplotype A G A C (SNP2-V27I-E114G-SNP1) associated with c.235delC was found [7]. In addition, the age of c.235delC (~11,500 years) estimated by one distant SNP marker (SNP6 or rs747931) was calculated [7]. It is worth noting that this particular SNP differentiated the haplotypes found in our study (G A C T and G A C C, underlined) (Table S2, Figure 6). Assuming a probable recombination of this distant SNP (rs747931) and a potential effect of genetic drift, our data on the structure of variable haplotypes in North Asians may be consistent with the hypothesis proposed by Yan et al. (2003) about a single origin of c.235delC in the Baikal Lake region and its spreading to Mongolia, China, Korea and Japan through subsequent migrations [7]. However, in recent studies, a "younger" age of c.235delC was determined: ~6500 years and ~1125-3150 years in the

Conclusions
In this study, the comprehensive analysis of the GJB2 gene was performed among patients with HI of different ethnicities living in the Baikal Lake region in the Siberian part of Russia. We found that the contribution of the GJB2 gene variants to the etiology of hearing loss in Buryat patients is significantly lower (5.1%) than in Russian patients (28.9%). Further extensive studies (including the NGS technology) are necessary to elucidate the unidentified genetic causes of hearing loss in patients examined in our study. Audiological analysis revealed that the genotype with c.-23+1G>A and c.516G>C variants may be associated with a milder phenotype than the genotypes homozygous for the c.35delG variant. The haplotype analysis in chromosomes with c.-23+1G>A and c.35delG performed in comparison with available published data supports a major role of the founder effect in the expansion of these variants around the world. The diversity of the c.235delC haplotypes in Northern Asians may indicate an independent as well as the common origin of this GJB2 pathogenic variant in Asia. We believe that subsequent studies of the structure of haplotypes bearing c.235delC will elucidate the origin of this GJB2 pathogenic variant.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14051001/s1, Chapter S1: Sanger sequencing of identified 14 variants of the GJB2 gene in 165 patients with HI in the Republic of Buryatia; Chapter S2: Pathogenicity analysis in silico of c.-254C>T and c.-49G>A variants of the GJB2 gene; Chapter S3: Haplotype analysis of the c.-23+1G>A, c.35delG and c.235delC pathogenic variants in the GJB2 gene; Figure S1: The variants identified in the non-coding regions of the GJB2 gene; Figure S2: The variants identified in the coding region (exon 2) of the GJB2 gene; Figure S3: Ensemble centroid structure diagram for two variants, c.-254C>T and c.-49G>A, of the GJB2 gene, generated in the Sfold software (https://sfold.wadsworth.org/cgi-bin/srna.pl); Figure S4: The c.235delC-haplotypes frequency in Eastern Asia and Northern Asia; Table S1: Detection and primer sequences of 12 SNP-markers, flanking the region of 84536 kb, around the GJB2 gene in chromosome 13; and Table S2

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.